HCN, a triple-resonance NMR technique for selective observation of histidine and tryptophan side chains in ¹³C/¹⁵N-labeled proteins

HCN, a new 3D NMR technique for stepwise coherence transfer from ¹H to ¹³C to ¹⁵N and reverse through direct spin couplings ¹J_CH and ¹J_CH, is presented as a method for detection and assignment of histidine and tryptophan side-chain ¹H, ¹³C, and ¹⁵N resonances in uniformly ¹³C/¹⁵N-labeled proteins. Product-operator calculations of cross-peak volumes vs adjustable delay τ₃ were employed for determination of optimal τ₃. For the phosphatidylinositol 3-kinase (PI3K SH3 domain, MW = 9.6 kD ) at pH 6, H(C)N, the ¹H/¹⁵N projection, produced observable cross peaks within 20 min. and was completely selective for the single tryptophan and single histidine. The 3D HCN experiment yielded welldefined cross peaks in 20 h for the ¹³C/¹⁵N-labeled origin-specific DNA binding domain from simian virus 40 T-antigen (T-ag-OBD_131-259, MW = 15.4 kD) at pH 5.5. Resonances from all six histidines in T-ag-OBD were observed, and 11 of the 12 ¹H and ¹³C chemical shifts and 10 of the 12 ¹⁵N chemical shifts were determined. The ¹³C dimension proved essential in assignment of the multiply overlapping ¹H and ¹⁵N resonances. From the spectra recorded at a single pH, three of the imidazoles were essentially neutral and the other three were partially protonated (22-37%). HCN yielded strong cross peaks after 18 h on a 2.0 mM sample of phenylmethanesulfonyl fluoride (PMSF)-inhibited α-lytic protease (MW = 19.8 kD ) at pH 4.4. No spectra have been obtained, however, of native or boronic acid-inhibited α-lytic protease after 18 h at various temperatures ranging from 5 to 55°C, probably due to efficient relaxation of active-site imidazole ¹H and/or ¹⁵N nuclei.